Little genetic variability in resilience among cattle exists for a range of performance traits across herds in Ireland differing in Fasciola hepatica prevalence.

It is anticipated that in the future, livestock will be exposed to a greater risk of infection from parasitic diseases. Therefore, future breeding strategies for livestock, which are generally long-term strategies for change, should target animals adaptable to environments with a high parasitic load. Covariance components were estimated in the present study for a selection of dairy and beef performance traits over herd-years differing in Fasciola hepatica load using random regression sire models. Herd-year prevalence of F. hepatica was determined by using F. hepatica-damaged liver phenotypes which were recorded in abattoirs nationally. The data analyzed consisted up to 83,821 lactation records from dairy cows for a range of milk production and fertility traits, as well as 105,054 young animals with carcass-related information obtained at slaughter. Reaction norms for individual sires were derived from the random regression coefficients. The heritability and additive genetic standard deviations for all traits analyzed remained relatively constant as herd-year F. hepatica prevalence gradient increased up to a prevalence level of 0.7; although there was a large increase in heritability and additive genetic standard deviation for milk and fertility traits in the observed F. hepatica prevalence levels >0.7, only 5% of the data existed in herd-year prevalence levels >0.7. Very little rescaling, therefore, exists across differing herd-year F. hepatica prevalence levels. Within-trait genetic correlations among the performance traits across different herd-year F. hepatica prevalence levels were less than unity for all traits. Nevertheless, within-trait genetic correlations for milk production and carcass traits were all >0.8 for F. hepatica prevalence levels between 0.2 and 0.8. The lowest estimate of within-trait genetic correlations for the different fertility traits ranged from -0.03 (SE = 1.09) in age of first calving to 0.54 (SE = 0.22) for calving to first service interval. Therefore, there was reranking of sires for fertility traits across different F. hepatica prevalence levels. In conclusion, there was little or no genetic variability in sensitivity to F. hepatica prevalence levels among cattle for milk production and carcass traits. But, some genetic variability in sensitivity among dairy cows did exist for fertility traits measured across herds differing in F. hepatica prevalence.

Evaluation of modified Dennis parasitological technique for diagnosis of bovine fascioliasis.

INTRODUCTION: Bovine fascioliasis causes important economic losses, estimated at COP$ 12,483 billion per year; its prevalence is 25% in dairy cattle. Parasitological techniques are required for it diagnosis. The Dennis technique, modified in 2002, is the one used in Colombia, but its sensitivity, specificity and validity are not known.  OBJECTIVE: To evaluate the validity and performance of the modified Dennis technique for diagnosis of bovine fascioliasis using as reference test the observation of parasites in the liver.  MATERIALS AND METHODS: We conducted a diagnostic evaluation study. We selected a convenience sample of discarded bovines sacrificed between March and June, 2013, in Frigocolanta for the study. We collected 25 g of feces from each animal and their liver and bile ducts were examined for Fasciola hepatica. The sensitivity, specificity, predictive positive value, predictive negative value, and validity index were calculated with 95% confidence intervals. The post-mortem evaluation was used as the gold standard.  RESULTS: We analyzed 180 bovines. The sensitivity and specificity of the modified Dennis technique were 73.2% (95% CI=58.4% - 87.9%) and 84.2% (95% CI= 77.7% - 90.6%), respectively. The positive predictive value was 57.7% (95% CI= 43.3% - 72.1%) and the negative one 91.4% (95% CI= 86.2% - 96.6%). The prevalence of bovine fascioliasis was 22.8% (95% CI= 16.4% - 29.2%).  CONCLUSION: The validity and the performance of the modified Dennis technique were higher than those of the traditional one, which makes it a good screening test for diagnosing fascioliasis for population and prevalence studies and during animal health campaigns.

Study of surface carbohydrates in Galba truncatula tissues before and after infection with Fasciola hepatica.

The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed.

Study of surface carbohydrates in Galba truncatula tissues before and after infection with Fasciola hepatica

The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed.

People Participation Towards Opisthorchis viverrini Prevention and Control in Chaiyaphum Province, Northeastern Thailand.

This cross-sectional descriptive and qualitative study was aimed to study the people participation and their approaches toward the human carcinogenic liver fluke, Opisthorchis viverrini, prevention and control in Ban Chaun sub-district administrative organization (BCSAO) and Bamnet Narong sub-district municipality (BNSM), Bamnet Narong district, Chaiyaphum Province, Thailand between June 2013 and February 2014. Participants were purposive selected, included chiefs of sub-district administrative organizations, sub-district municipalities, sub-district health promotion hospitals (SHPHs), heads of village, and a further sample was selected with a multi- stage random sampling for public health volunteers, and villagers. The pre-designed questionnaire contained items for individualized status and the participatory steps of sharing ideas, decision-making, and planning, procession, evaluation, and mutual benefit, for the project O. viverrini prevention and control (POPC). In-depth interviews were used for collection of need approaches to POPC. With 375 participants who completed the questionnaire, it was found that people had a high level regarding to participate in the POPC, particularly in the process stage (X_ =3.78, S.D. = 0.56), but the lowest level was found in sharing ideas, decision making, and planning step (X_ =3.65, S.D. = 0.63). By comparison, participant status and organization did not significantly differ with people participation. In each step, Ban Chaun sub-district had a high level of participation in the step of sharing ideas, decision making, and planning toward POPC, more than Bamnet Narong sub-district municipality (t=2.20, p=0.028). Approaches for POPC in Ban Chaun sub-district and Bamnet Narong sub-district municipality included requirements for budget support, annual campaigns for liver fluke prevention and control, campaign promotion, risk group observation, home visiting, community rules regarding reducing raw fish consumption in their communities, and a professional public health officer for working in their communities, BCSAO, BNSM, and SHPH, for O. viverrini prevention and control. This study indicates that people realize that eradication of the liver fluke needs a continuous people participation for O. viverrini prevention and control in their communities.

Fasciolosis aguda: presentación de un caso de Cuba / Acute fasciolosis: a Cuban case report

Fasciolosis es una enfermedad parasitaria de humanos y diferentes animales. Sus agentes causales son Fasciola hepatica y Fasciola gigantica; sin embargo, en Cuba, solo la primera de esas especies ha sido notificada. Como otras enfermedades incorporadas en el grupo de las olvidadas o desatendidas, fasciolosis, es más frecuente en personas que viven en áreas rurales donde coexisten animales infectados, hospederos intermediarios y hospederos susceptibles. Se exponen las características de un caso que pudiera contribuir en el proceso de educación continuada de los profesionales de la salud en nuestra área geográfica(AU)

Fasciolosis is a worldwide parasitic disease in human and domestic animals. The causative agents of fasciolosis are Fasciola hepatica and Fasciola gigantica but, in Cuba, only F. hepatica has been notified. As others neglected diseases, F. hepatica is more common in people living in rural areas where infected animals, intermediate hosts and susceptible hosts coexist. We describe the clinical features of a case that could contribute to the process of continuing medical education of health professionals in our geographic area(AU)

Role of the apparent diffusion coefficient measurement by diffusion weighted magnetic resonance imaging in the diagnosis of Fasciola hepatica in the liver.

PURPOSE: The aim of this study was to investigate the diagnostic role of the apparent diffusion coefficient (ADC) measurement in the diagnosis of focal parenchymal lesions and to understand the discriminating role of the ADC value for differentiating Fasciola lesions from other focal liver lesions. MATERIALS AND METHODS: We measured ADC values of parenchymal lesions and liver parenchyma in 18 patients with Fasciola hepatica infestation at b 100, b 600, and b 1000 s/mm(2) gradients. We further measured average ADC values of hepatic metastases (n = 21), hepatocellular carcinomas (n = 21), cholangiocarcinomas (n = 7), hydatid cysts (n = 12), and focal nodular hyperplasia (FNH) (n = 12) and compared them with average ADC values for Fasciola hepatica. RESULTS: The differences between average ADC values of lesions (2.16 ± 0.36 × 10(-3) mm(2)/s) and parenchyma (1.64 ± 0.2 × 10(-3) mm(2)/s) at three gradients were statistically significant (P < 0.05). Mean ADC values of Fasciola hepatica lesions were significantly different from most of the other focal hepatic lesions, except FNH at all gradients and hydatid cyst at only the b 100 gradient. CONCLUSION: ADC measurement may be a complementary method in the diagnosis of Fasciola hepatica, and it may be used to differentiate these lesions from other focal liver lesions.

Towards assessing fine-scale indicators for the spatial transmission risk of Fasciola hepatica in cattle.

In order to improve the spatial resolution of current risk maps for fasciolosis in cattle, more knowledge is needed with respect to farm-level factors that determine infection risk. In this study, we visited 39 dairy farms within a predefined low- and high-risk area for fasciolosis in Belgium and assessed their infection status by an indirect bulk tank milk enzyme-linked immunosorbent assay (ELISA). Management factors were collected and all pastured lands of the farms were visited to identify and georeference potential snail habitats. The habitats were visually characterised, investigated for the presence of the intermediate host snails of Fasciola hepatica (i.e. Galba truncatula and Radix spp) and used in a geographical information system (GIS) to construct overlays including information on soil and hydrology. A linear regression model was used to evaluate associations between bulk tank milk ELISA results and farm level management and habitat factors. A logistic, mixed model was used to identify possible risk factors for the presence of intermediate host snails on a potential habitat. Potential snail habitats were found in 35 out of 39 farms. A total of 87 potential habitats were identified and on 29% of these, intermediate host snails were found. The number of potential habitats, the presence of snails, drainage of pastures, month of turnout of the cows, stocking rate, type of watering place and risk area were significantly associated with the bulk tank milk ELISA result and explained 85% of the observed variation. Intermediate host snails were more likely to be present with increasing surface of the potential habitat and on loamy soils. This study confirms the importance of farm management factors in the infection risk for F. hepatica in cattle and highlights that the combination of management factors with characterization of snail habitats is a powerful means to predict the infection risk with F. hepatica at the individual farm level. Further research is needed to investigate how this knowledge can be incorporated in nation-wide spatial distribution models of the parasite.

Laparoscopy-assisted right hepatectomy in a case of Fasciola hepatica.

Laparoscopic hepatectomy provides the usual advantages of a minimally invasive surgery. This study presents a case of Fasciola hepatica infection that was successfully treated with laparoscopic hepatectomy. The patient was referred because of persistent fever and right hypochondralgia with a huge mass occupying the right lobe of the liver, which was detected by imaging analysis. Serologic tests indicated an F. hepatica infection. The patient underwent a laparoscopic resection because the anthelmintic agent, triclabendazole was not effective. During the surgical technique, 5 trocars were inserted. After liver mobilization, the Glissonian pedicles and right hepatic veins were safely taped. A Penrose drain was placed behind the liver for a liver-hanging maneuver. A liver parenchymal transection was performed through an 8 cm handport site using a dissecting sealer (TissueLink Medical) after precoagulating its superficial layer by microtaze. Glissonian pedicles and the right hepatic vein were divided using an endolinear stapler (endcutter 45, Ethicon). Finally, the resected specimen was extracted from the handport. The surgical time was 450 minutes and the surgical blood loss was 370 mL. The patient was discharged 10 days after the surgery with an uneventful postoperative course. The laparoscopy-assisted hepatectomy in this case was beneficial for the patient's quality of life as a minimally invasive operation with a high degree of safety.

Conservación de heces humanas para la detección de antígenos de excreción-secreción de Fasciola hepatica / Preservation of human stools for the detection of Fasciola hepatica excretory secretory antigens

INTRODUCCIÓN: la fasciolosis en Cuba es una enfermedad endémica en el ganado y en los últimos años ha tenido un incremento en el número de casos reportados en humanos. OBJETIVOS: por causa de las dificultades para realizar el diagnóstico de esta enfermedad en zonas rurales o alejadas del laboratorio se ha hecho necesario buscar soluciones preservantes, que permitan conservar las muestras de heces hasta el momento de la determinación de antígeno por el método de FasciDIG®. MÉTODOS: se utilizaron diferentes soluciones preservantes (dicromato de potasio 2 por ciento, hibitane acuoso 0,5 por ciento, glutaral 2 por ciento, cloruro de benzalconio 1 por ciento, azida sódica 0,04 por ciento y agua destilada con tween 20 0,05 por ciento) a temperatura ambiente, 4 ºC y - 20 ºC. RESULTADOS: en las muestras preservadas a temperatura ambiente, las que no tenían preservo sufrieron un gran deterioro, mostraron valores superiores a las conservadas con agua tween 20 y con azida sódica. Esta última es un producto altamente tóxico, por ser un inhibidor de la cadena respiratoria y requeriría de un mayor cuidado en su utilización. CONCLUSIONES: se sugiere que la solución preservante de agua destilada con tween 20 0,05 por ciento a temperatura ambiente mostró los mejores resultados en el estudio(AU)

INTRODUCTION: fascioliasis is an endemic disease in cattle in Cuba, but in the last few years, there has been an increase in the number of reported human cases. OBJECTIVES: the difficulties in diagnosing this disease in rural areas or in zones far away from the laboratory make it necessary to find adequate solutions that allow preserving the stool specimens until the antigen is detected by the FasciDIG® method. METHODS: several preserving solutions were used (2 percent potassium dichromate, 0,5 percent aqueous hibitane, 2 percent glutaral, 1 percent benzalkonium chloride, 0,04 percent sodic azide and 0,05 percent distilled water with 20 tween) at room temperature, 4 ºC and - 20 ºC. RESULTS: among the samples kept at room temperature, those with no preserve suffered a significant deterioration, and their values were higher than the ones of the samples conserved with distilled water with tween 20 and with sodium azide. The latter is a very toxic product and its use would require a greater care. CONCLUSIONS: 0,05 percent distilled water with 20 tween at room temperature was the preserving solution with the best results in this study(AU)

Parasite development and visceral pathology in Galba truncatula co-infected with Fasciola hepatica and Paramphistomum daubneyi.

Histological investigations in Galba truncatula naturally or experimentally co-infected with Fasciola hepatica and Paramphistomum daubneyi were carried out to study parasite development and the responses of the digestive gland and kidney of snails, as larval forms of these digeneans often use these two sites for their growth within the snail's body. The number of live rediae per snail ranged from 2.4 to 4.2 for the dominating parasite (it developed in the digestive gland) and was less than 2.0 for the other species. When the dominating species was F. hepatica, most snails harboured cercariae-containing rediae; if this parasite was P. daubneyi, procercariae-containing rediae with or without free procercariae were observed in most snails. In contrast, most rediae of the other species were immature. The pathology caused by the dominating species in the digestive gland was greater than that recorded in the kidney, where the other parasite was generally located. The most frequent tissue lesions in the digestive gland were generalized epithelial necrosis and epithelial reconstitution. In the kidney, multifocal epithelial necrosis was frequently observed, particularly when P. daubneyi was the dominating species. The frequencies of lesions in the digestive gland agreed with percentages reported by our team in other snails mono-infected with F. hepatica or P. daubneyi. In contrast, multifocal necrosis in the kidney was clearly greater in the present study and this finding might be explained by assuming that a sufficient number of free larvae within the snail would be necessary for the development of epithelial necrosis in the whole kidney.

Hospederos intermediarios de fasciola hepática en el Perú / Intermediate hosts of in Peru fasciola hepatica

Se demostró la infección natural y experimental de los caracoles limneídos peruanos con estadios larvales de Fasciola hepatica. Los índices de infección natural y experimental fueron del 12% y 28% en Lymnaea columella y del 27% y 70% en Lymnaea viatrix, respectivamente. Lymnaea diaphana y Lymnaea cousini no presentaron infección natural y demostraron no ser susceptibles a la infección experimental.

The natural and experimental infection of Peruvian lymnaeid snails with larval stages of Fasciola hepatica was demonstrated. The index of natural and experimental infection was of 12% and 28% in Lymnaea columella and of 27% and 70% in Lymnaea viatrix, respectively. Lymnaea diaphana and Lymnaea cousini did not present natural infection and demonstrated not to be prone to experimental infection.

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite.

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.

Remote sensing and climate data as a key for understanding fasciolosis transmission in the Andes: review and update of an ongoing interdisciplinary project.

Fasciolosis caused by Fasciola hepatica in various South American countries located on the slopes of the Andes has been recognized as an important public health problem. However, the importance of this zoonotic hepatic parasite was neglected until the last decade. Countries such as Peru and Bolivia are considered to be hyperendemic areas for human and animal fasciolosis, and other countries such as Chile, Ecuador, Colombia and Venezuela are also affected. At the beginning of the 1990s a multidisciplinary project was launched with the aim to shed light on the problems related to this parasitic disease in the Northern Bolivian Altiplano. A few years later, a geographic information system (GIS) was incorporated into this multidisciplinary project analysing the epidemiology of human and animal fasciolosis in this South American Andean region. Various GIS projects were developed in some Andean regions using climatic data, climatic forecast indices and remote sensing data. Step by step, all these GIS projects concerning the forecast of the fasciolosis transmission risk in the Andean mountain range were revised and in some cases updated taking into account new data. The first of these projects was developed on a regional scale for the central Chilean regions and the proposed model was validated on a local scale in the Northern Bolivian Altiplano. This validated mixed model, based on both fasciolosis climatic forecast indices and normalized difference vegetation index values from Advanced Very High Resolution Radiometer satellite sensor, was extrapolated to other human and/or animal endemic areas of Peru and Ecuador. The resulting fasciolosis risk maps make it possible to show the known human endemic areas of, mainly, the Peruvian Altiplano, Cajamarca and Mantaro Peruvian valleys, and some valleys of the Ecuadorian Cotopaxi province. Nevertheless, more climate and remote sensing data, as well as more accurate epidemiological reports, have to be incorporated into these GIS projects, which should be considered the key in understanding fasciolosis transmission in the Andes.

Íctero obstructivo, Fasciola hepatica: presentación de un nuevo caso / Obstructive jaundice, Fasciola hepatica: a new case report

Se reportó un caso de íctero obstructivo provocado por Fasciola hepatica en una mujer blanca de 35 años. El diagnóstico es efectuado por colangiopancreatografía retrógrada endoscópica y corroborado por la presencia de huevos del parásito en las heces fecales. Recibió tratamiento con triclabendazol, lográndose mejoría del cuadro clínico y negativización de las copas cónicas en los controles seriados realizados a los 7, 30, 60 y 90 d posterapéutica(AU)

Emergence of Opisthorchis viverrini cercariae from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos.

Under natural conditions, the emergence of Opisthorchis viverrini cercariae from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos showed diurnal periodicity, peaking between 8:00-10:00 AM. The cercariae did not emerge during darkness, but low-intensity light could induce a release. Cercariae shedded from each field infected B.(D.) s. goniomphalos was recorded daily. The maximum output from one snail was 1,728 cercariae in a day. The total cercarial output from all five infected snails was 56,555 and the maximum of total cercariae shed from one snail was 27,692. The field-infected B. (D.) s. goniomphalos could survive for 70 days after the snails were collected.

The molluscicidal activities of some Euphorbia milii hybrids against the snail Indoplanorbis exustus.

The objective of this study was to observe the molluscicidal activities of Euphorbia milli, known as "poysean" in Thailand, against Indoplanorbis exustus. Latex from 12 different E. milii hybrids was screened for its molluscicidal activities. Indoplanorbis exustus were exposed for 24 and 48 hours to the latex at various concentrations ranging from 6 to 25 ppm and mortality rates were recorded. Eight hybrids of latex were effective. The six most effective hybrids were E. milii Dang-udom, E. milii Arunroong, E. milii Raweechotchuong, E. milii Srisompote, E. milii Sri-umporn and E. milii Tongnopakun, which killed all snails after 24 hours of exposure. Under the same conditions, latex of E. milii Dowpraket and E. milii Promsatid killed 50% of the snails. Such results indicate that these 6 hybrids seem promising as natural molluscicidal agents.

Uso do Sm14 recombinante (sm14r) na vacinação contra infecção experimental por Fasciola hepatica em coelhos

A proteína Sm14, Sm de Schistosoma mansoni e 14 representando o peso molecular aproximado em kDa, que pertence a família das proteínas que se ligam aos ácidos graxos (fatty acid binding proteins – FABP) é considerada um dos candidatos à vacina contra esquistossomose humana. Dado o grau de homologia entre a Sm14 e a Fh15 (fração de baixo peso molecular isolada da Fasciola hepatica), a sugestão lógica foi avaliar a eficácia imunogênica do antígeno recombinante, Sm14r, na infecção experimental por F. hepatica. Coelhos Nova Zelândia (NZ) foram imunizados com 80 microgramas do Sm14r e posteriormente desafiados com 40 metacercárias de F. hepatica. Foram utilizadas duas amostras do Sm14r sendo que o lote 1 foi expresso no vetor pRSET A (Sm14r – 1) e o lote 2 no vetor pAE (Sm14r – 2). As seqüências de aminoácidos dos lotes utilizados neste estudo possuem o aminoácido treonina na posição 20 (Sm14-T20). Os ensaios imunoenzimáticos (ELISA) para detecção de anticorpos IgG contra os antígenos recombinantes revelaram que os grupos de animais imunizados com as proteínas associadas ao adjuvante (Sm14r – 1 + ACF e Sm14r – 2 + ACF) apresentaram níveis altos e significativos de anticorpos até o final do experimento. A produção de anticorpos anti-F. hepatica em coelhos NZ, experimentalmente infectados com esse trematódeo, foi acompanhada através da técnica de ELISA, utilizando o antígeno SOM (antígeno somático de vermes adultos de F. hepatica) e SE (extrato salino de F. hepatica). Anticorpos IgG anti-SOM e anti-SE foram detectados no período de 30 dias após a infecção

Paralelamente foram realizados exames coproparasitológicos individuais, sendo os primeiros ovos detectados nas fezes 90 dias após a infecção. Cento e trinta dias após a infecção, os animais foram necropsiados para contagem dos vermes de F. hepatica. Houve uma total correlação entre os níveis de anticorpos anti-SOM detectados por ELISA nos soros dos animais infectados, presença de ovos nas fezes, recuperação de vermes e danos hepáticos (através de análises macroscópica e microscópica). Com o objetivo de estudar o perfil imunofenotípico dos leucócitos circulantes do sangue periférico de coelhos imunizados e desafiados com metacercárias de F. hepatica (avaliação da resposta imune no contexto ex vivo) foram utilizados anticorpos monoclonais usados em ensaios de citometria de fluxo: CD4, CD8, CD44, CD18 e CD25. Os resultados permitiram observar que o protocolo vacinal onde houve associação das proteínas com o adjuvante favoreceu um maior número de alterações imunológicas. As proteínas Sm14r – 1 e Sm14r – 2 se mostraram antigênicas, mas no entanto, não foi possível avaliar a atividade protetora dos antígenos recombinantes através da redução da carga parasitária e danos hepáticos pela falta de viabilidade das metacercárias utilizadas neste estudo